EMBO/SourceData huggingface dataset:
Figure 6A Subcellular fractionation of BcSrpk1-GFP in WT and BcSrpk1mutant strains. The amount of nuclear BcSrpk1-GFP was set as 1. Data information: , each gel shown is a representative experiment carried out three times. The fractionation controls were histone H1 (nucleus), Actin (cytosol) and CBB staining.B Co-IP analysis revealing the interaction of BcAha1 with BcSrpk1 and its mutant forms. Data information: each gel shown is a representative experiment carried out three times.C K367 acetylation of BcSrpk1 during the early infection stage. Total proteins were extracted from tomato leaves at 0 (spores), 12, 24 and 36 hpi. Eluted proteins bound to the anti-GFP beads were probed by the anti-K304ac and anti-GFP antibodies. The amount of acetylated and total BcSrpk1 at 0 hpi was each set as 1. Data information: each gel shown is a representative experiment carried out three times.F Relative density of DAB staining in E (four biological replicates). Data information: For F the presence of different letters above the mean values of four replicates indicates a significant difference between different samples (P < 0.05, ANOVA). The whiskers of the boxplots indicate the upper and lower quartiles, the boxes indicate the interquartile range, and the plus sign indicates the mean.
Figure 1Presence of TOC proteins in the central vacuole of protoplasts. Arabidopsis protoplasts expressing the YFP-tagged chloroplast proteins were analyzed by confocal microscopy following ConA treatment for four hours. Representative images are shown (A). Chlorophyll autofluorescence was used to determine the localization of the YFP fluorescence signals relative to the chloroplasts. Signals for SP1-YFP and FAX1-YFP, which here are control proteins, were only present at the chloroplast envelope. Accumulation of YFP puncta in the central vacuole is indicated by the arrowheads. Scale bar, 10 μm.Presence of TOC proteins in the central vacuole of Arabidopsis leaf and hypocotyl cells. Eight-day-old transgenic Arabidopsis seedlings stably expressing pToc33:YFP-Toc33 or pToc75:YFP-Toc75 were treated with ConA for two days, and then analyzed by confocal microscopy. Accumulation of YFP puncta in the central vacuole is indicated by the arrowheads. Scale bar, 10 μm.
Figure 2Lack of TOC proteins in the central vacuole of atg7-2 mutant hypocotyl cells. Eight-day-old transgenic Arabidopsis seedlings stably expressing pToc33:YFP-Toc33 and pToc75:YFP-Toc75 in the atg7-2 mutant background were treated with ConA for two days, and then analyzed by confocal microscopy. Note that YFP-Toc33 and YFP-Toc75 bodies (arrowheads) were retained at plastid bridges and extensions, and not released into the vacuole, as occurs in the WT background Scale bar, 10 μm.Autophagy inhibition affected accumulation of TOC proteins. Eight-day-old wild-type (WT) and atg7-2 plants were treated respectively with proteasome inhibitor, bortezomib (Btz), cysteine protease inhibitor, E64d, or DMSO (Mock) for one day. Total protein extracts from these plants were analyzed by immunoblotting using antibodies as indicated. Actin acted as a loading control. Typical immunoblotting results are shown (B). In B, the arrowhead indicates full-length Toc159, and the hollow arrow indicates a truncated form of Toc159. Molecular weight markers (sizes in kD) are indicated to the right of the images.Autophagy inhibition affected accumulation of TOC proteins. Eight-day-old wild-type (WT) and atg7-2 plants were treated respectively with proteasome inhibitor, bortezomib (Btz), cysteine protease inhibitor, E64d, or DMSO (Mock) for one day. Band intensities were quantified and normalized to equivalent data for Actin (C). Data are means ± SEM from three biological replicates. Asterisks in C indicate significance according to an unpaired two-tailed Student's t-test (*, significant at P < 0.05; **, significant at P < 0.01).
Figure 3Presence of autophagosome in the chloroplast envelope. Arabidopsis protoplasts expressing free YFP and YFP-ATG8 (autophagosome marker) were analyzed by confocal microscopy. Corresponding chlorophyll autofluorescence images were employed to orientate the YFP signal in relation to the chloroplasts. For eliminating interference from cytosolic signals, intact protoplasts (upper panels) were ruptured using 10-μm nylon mesh to isolate the chloroplasts. Retention of YFP signals in association with chloroplasts upon rupturing the cell (lower panels) clearly indicated that the YFP-ATG8 was associated with chloroplasts. Representative images are shown (A).whole protein samples of protoplasts and purified chloroplasts were analyzed by SDS-PAGE and immunoblotting, using antibodies against the YFP tag and Tic110 (a chloroplast marker). Typical immunoblotting results are shown (B).Presence of NBR1-HA in chloroplast fraction. The protoplasts were isolated from four-week-old transgenic Arabidopsis plants stably expressing p35S:NBR1-HA (NBR1-HA) and p35S:YFP-HA (YFP-HA), followed by chloroplast purification, respectively. Protein samples of whole protoplasts and isolated chloroplasts of NBR1-HA and YFP-HA were then analyzed by SDS-PAGE and immunoblotting, using antibodies against the HA tag and Tic40. Typical immunoblotting results are shown (G).
Figure 4BiFC results showed interaction between NBR1 and TOC proteins. cYFP and nYFP refer to C-terminal YFP fragment and N-terminal YFP fragment, respectively. Reconstitution of YFP fluorescence was assessed after transient coexpression of the indicated pairs of fusion proteins. CDKA1 and OEP7 were used as negative controls for OEM and cytosolic proteins, respectively. Representative images are shown. Scale bars, 10 μm.­ The frequency of protoplasts showing a BiFC signal in C was quantified. All data are means ± SEM from three experiments.NBR1 coimmunoprecipitated with TOC proteins. Proteins were extracted from eight­-day-old transgenic Arabidopsis seedlings stably expressing p35S:NBR1-HA or p35S: NBR1-ΔUBA-HA, or control seedlings only expressing p35S:YFP-HA, following UVB treatment for four hours. Obtained protein extract was analyzed by IP using anti-HA resin, and analyzed by immunoblotting of four endogenous proteins or HA-tagged proteins. Tic40 acted as negative controls. The asterisks indicate nonspecific cross-reacting bands. Arrow indicates the NBR1-HA band. The arrowhead indicates full-length Toc159 protein. Molecular weight markers (sizes in kD) are indicated to the right of the images.
Figure 5Phenotypes of seedlings under UVB stress. Briefly, eight-day-old WT, atg7-2, nbr1-2 and NBR1-OX (overexpressor) seedlings grown under normal condition were subjected to UVB stress, with UVB treatment for 3 hours and recovery for 7 days. Representative plant images (A) and total chlorophyll content values (B) are shown; the latter provide a convenient metric for biomass. The chlorophyll values for the mutants and overexpressor plants were statistically significantly different from that for the WT (Student's t test; *, significant at P < 0.05; ***, significant at P < 0.001).TOC protein levels were regulated by autophagy in response to short-term UVB stress. Eight-day-old plants of indicated genotypes were subjected to UVB stress for the indicated times, and then analyzed by immunoblotting using 10 μg total protein extract loaded per lane (C, upper). Coomassie staining indicated that the treatments did not change the overall protein profile (C, lower). Band intensities were quantified and normalized to equivalent Actin data (D) Time 0 was taken as 100%. Unpaired two-tailed Student's t-tests revealed that the Toc159 protein level in nbr1-2 (P < 0.05) and NBR1-OX (P < 0.05), the Toc75 protein level in atg7-2 (P < 0.05), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.05), and the Toc33 protein level in atg7-2 (P < 0.05), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.05), were significantly different from levels in the WT at the time of 1 d recovery from UVB stress.Toc159 was modified by both K48 and K63 ubiquitin linkage types. Eight-day-old WT plants were grown under normal growth condition (-), or subjected to UVB stress (+), with UVB treatment for four hours. The protein extract from the plants was analyzed by IP using native Toc159 antibody, or its preimmune serum as a negative control, and analyzed by immunoblotting using antibodies as indicated to the left of the images (E). Tic40 acted as a negative control. The asterisk indicates a truncated Toc159 form without A domain Molecular weight markers (sizes in kD) are indicated to the right of the images. Please note that ubiquitin antibodies tend to produce stronger signals for hyper-ubiquitinated species than for hypo-ubiquitinated or mono-ubiquitinated species (based on the number of epitopes), which contrasts with the behaviour of the Toc159 antibody. Thus, the signals of the two antibodies are not expected to fully overlap. Poly-ubiquitinated Toc159 may not be detected by the Toc159 antibody due to the much lower abundance of these species compared with the mono-ubiquitinated or unmodified forms. This is why the poly-Ub K48 and K63 signals appear at higher molecular weight positions than the Toc159 signals. Relative Toc159 amounts modified by K48 or K63 ubiquitin linkage in plants with or without UVB stress were assessed by quantifying the smeary band intensity of the IP samples in α-K48 or α-K63 blots (F). All data are means ± SEM from three experiments.
Figure 6TOC protein levels were regulated by autophagy in response to short-term heat stress. Eight-day-old plants of indicated genotypes grown in vitro were subjected to short term heat stress, recovered for the indicated times, and then analyzed by immunoblotting using 10 μg total protein extract loaded per lane (B, upper). Coomassie staining indicated that the treatments did not change the overall protein profile (B, lower). Band intensities were quantified and normalized to equivalent Actin data (C). Time 0 was taken as 100%. Data are means ± SEM from three biological replicates. Unpaired two-tailed Student's t-tests revealed that the Toc159 protein level in nbr1-2 (P < 0.05) and NBR1-OX (P < 0.01), and the Toc75 protein level in NBR1-OX (P < 0.05), were significantly different from levels in the WT at Time 12 h; and that the Toc159 protein level in atg7-2 (P < 0.001), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.001), and the Toc75 protein level in atg7-2 (P < 0.01), nbr1-2 (P < 0.01), and NBR1-OX (P < 0.01), were significantly different from levels in the WT at Time 24 h.
Figure 7Analysis of protein import efficiency in vitro. Chloroplasts were isolated from 12-day-old WT, nbr1-2 and NBR1-OX seedlings grown in vitro under normal condition, and were subjected to in vitro protein import assay by using 35S-labeled Rubisco small subunit (SSU) precursor protein as the import substrate, followed by corresponding quantification of the maturation of the radiolabeled precursor protein. A representative phosphor screen image is shown (upper left), and Coomassie staining of Rubisco large subunit (LSU) indicated equal loading (lower left); Times indicate minutes after the start of each import reaction. IVT, in vitro translation product used as a control. Together with similar images from two additional experiments, this was used to conduct the quantitative analysis shown (right); the amount of imported, mature protein in each sample was expressed as a percentage of that present at the final time point for the WT. Data are means ± SEM from three biological replicates.Chloroplast development in WT, nbr1-2 and NBR1-OX seedlings under UVB stress was studied. Cotyledons of eight-day-old plants grown in vitro under normal condition (D) or UVB stress (E) were analyzed by transmission electron microscopy, and representative images are shown. UVB stress was conducted in the same procedures as in C. In D and E, the upper images are at the same magnification (scale bar, 2 μm); and below these are high magnification (5×) images corresponding to the boxed regions above. These and similar micrographs of chloroplasts were used to determine chloroplast shape (F), the number of thylakoid lamellae per granum (G), and the number of starch granules per chloroplast (H) in each genotype, with or without UVB stress. Asterisks indicate significance according to an unpaired two-tailed Student's t-test (*, significant at P < 0.05; ***, significant at P < 0.001; ****, significant at P < 0.0001). Values are means ± SEM from 30-50 chloroplasts.
Figure 8Phenotypes of two-week-old WT, nbr1-2, ppi1 and ppi1 nbr1-2 seedlings grown on soil. Representative plant images (A) and chlorophyll contents (B) are shown. The chlorophyll values for the ppi1 and ppi1 nbr1-2 plants were statistically significantly different (Student's t test; ****, significant at P < 0.0001). Values are means ± SEM from 20 plants.Chloroplast development in ppi1 and ppi1 nbr1-2 seedlings was studied. Cotyledons of two-week-old plants grown under normal condition as in A were analyzed by transmission electron microscopy, and representative images are shown (E). In E, the upper images are at the same magnification (scale bar, 1 μm); and below these are high magnification (3×) images corresponding to the boxed regions above. These and similar micrographs of chloroplasts were used to determine chloroplast size (F) and the number of thylakoid lamellae per granum (G) in each genotype. Asterisks indicate significance according to an unpaired two-tailed Student's t-test (****, significant at P < 0.0001). Values are means ± SEM from 30-40 chloroplasts.
Figure 1A) Image of 8-9 day old male control (w1118, left) and chmRNAi (right) flies upon ubiquitous chm knockdown (left) and the corresponding quantification (right) (N = 6, Paired t-test was performed for statistical significance); B) Image of 8-9 day old male control (w1118, left) and chmRNAi (right) flies upon fat-body specific chm knockdown (left) and the corresponding quantification (right) (N = 4, Paired t-test was performed for statistical significance); C) Image of 8-9 day old male control (w1118, left) and chmRNAi (right) flies upon neuron specific chm knockdown (left) and the corresponding quantification (right) (N = 3, Paired t-test was performed for statistical significance); D) Image of 8-9 day old male control (w1118, left) and chmRNAi (right) flies upon muscle specific chm knockdown (left) and the corresponding quantification (right) (N = 3, Paired t-test was performed for statistical significance) Data information: All replicates are independent biological replicates and errors bars indicate standard error of the mean (SEM). Paired t-test or Tukey test was performed as indicated and non-significant values are not shown (* - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001).
Figure 3A) Heatmap showing all significant proteins (FDR < 0.05 and Π < 0.05) between control (w1118) and chmRNAi. Values in the heatmap are scaled raw intensity values. NAs are indicated in grey;
Figure 4A) Average survival curve of control (w1118) and chmRNAi male flies upon ubiquitous knockdown of chm with da-Gal4 (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); B) Average survival curve of control (w1118) and chmRNAi male flies upon fatbody knockdown of chm with r4-Gal4 (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); C) Average survival curve of control (w1118) and chmRNAi male flies upon neuronal knockdown of chm with elav-Gal4 (N = 4, paired , Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); D) Average survival curve of control (w1118) and chmRNAi male flies upon muscle knockdown of chm with mef-Gal4 (N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001)); E, F Average survival curve between E) ethanol (N = 3, paired Log-rank test was performed for each biological replicate. p-values ranged between 0.2 to 0.05 across replicates) and F) RU486 (N = 4, paired, Log-rank test was performed for each biological replicate with all of them having a p-value < 0.05)) and administered control (w1118) and chmRNAi male flies using Act-GS-Gal4 for adult-specific chm knockdown; G) Average survival curve of control (w1118) and chmMYST/+ male flies (N = 5, paired Log-rank test was performed for each biological replicate with all of them having a p-value < 0.0001). Data information: All replicates are independent biological replicates and errors bars indicate standard error of the mean (SEM). For survival curves, log-rank test was performed for each biological replicate. The displayed p-value is based on all biological replicates.
Figure 5D-G Box plot (left) and heatmap (right) of annotated genes from GO terms of D) Cellular response to stress (ANOVA p-value: 6.49 x10-08); E) Response to starvation (ANOVA p-value: 3.09 x10-07); F) Carbohydrate metabolic process (ANOVA p-value: 5.46 x10-07); G) Lipid metabolic process (ANOVA p-value: 7.88 x10-09); Box plot shows the average vst normalized read counts of all annotated genes within the GO term averaged over the replicates. Boxes indicate inter-quartile range with central band as the median, central filled dot as the average and the whiskers indicating the maximum and minimum values across 5 biological replicates. Heatmap shows the scaled values of vst normalized read counts in each replicate for all conditions. Data information: All replicates are independent biological replicates and errors bars indicate standard error of the mean (SEM). Tukey test was performed and non-significant values are not shown (* - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001).
Figure 6A) Weight of 8-9 day old male chmMYST/+ and chmMYST/+;UAS-chmmyc flies upon weak (left, N = 5, paired) and strong (right, N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001) chm overexpression with arm-Gal4 and da-Gal4 respectively (Paired t-test was performed); Data information: All are independent biological replicates and errors bars indicate standard error of the mean (SEM). Paired t-test was performed for the fly weight data and non-significant values are not shown (* - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001).B) Average survival curve of chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon ubiquitous weak expression of chm with arm-Gal4 (N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001); C) Average survival curve of chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon ubiquitous strong expression of chm with da-Gal4 (N = 3, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001); Data information: All are independent biological replicates and errors bars indicate standard error of the mean (SEM). For survival curves, log-rank test was performed for each biological replicate. The displayed indicated p-value is based on all biological replicates. D) Average survival curve of chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon fatbody expression of chm with r4-Gal4 (N = 4, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001); E) Average survival curve of control chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon neuronal expression of chm with elav-Gal4 (N = 5, paired, Log-rank test was performed for each biological replicate. All replicates had p-values < 0.0001); F) Average survival curve of chmMYST/+ and chmMYST/+;UAS-chmmyc male flies upon muscle expression of chm with mef-Gal4 (N = 5, paired, Log-rank test was performed for each biological replicate. p-values ranged between 0.6 to 0.0001 across replicates). Data information: All are independent biological replicates and errors bars indicate standard error of the mean (SEM). For survival curves, log-rank test was performed for each biological replicate. The displayed indicated p-value is based on all biological replicates.
Figure 1(C) Fluorescence micrograph images of representative cells stained for NONO in KELLY cells treated with 2, 4, 6, 8 or 10% 1,6 hexanediol showing dissolution of NONO puncta with increasing concentration. (D) Dot plot of NONO foci area (µm2) per nucleus at different concentrations of 1,6 hexanediol as in (C). Bars are SD. The numbers of biological replicates are indicated in (C).(F) Fluorescence micrograph images of representative cells stained for NONO in KELLY cells treated with PBS, RNase A or DNase I, as indicated. (G) Dot plot of summed green fluorescence per nucleus in (F). Bars are SD. The numbers of biological replicates are indicated in (F).
Figure 2(A) Fluorescence micrograph images of representative cells stained for NONO and NEAT1_2 after transfection with plasmids expressing YFP fused NONO_WT and NONO_∆RRM1 protein. DAPI (blue) stain indicates cell nuclei, YFP fused NONO (green) and NEAT1_2 RNA FISH (red). (B) The enrichment of mean NONO fluorescence detected within RNA FISH foci is quantitatively determined as a ratio relative to mean nuclear NONO fluorescence in (A). Bars are SD. The numbers of biological replicates are indicated in (A).Sphericity per NONO segment, NONO foci number per nucleus and NONO foci size between YFP fused NONO_WT and NONO_∆RRM1 plasmids. Bars are SD. Biological replicates n=60 in (C) and n=50 in (D)
Figure 5(A) Relative NONO mRNA and protein levels in KELLY cells treated with control or NONO KD siRNAs. Representative Western blot image for NONO protein.
Figure 6(B) Western blot quantitation analysis of GATA2 and HAND2 protein levels between control and NONO KD siRNAs. (C) Representative Western blot images for GATA2 and HAND2 proteins in (B).
Figure 1C. qRT-PCR data showing induction of each gene in the iniBAC operon in WT Mtb cultured in low iron and in the virR mutant strain cultured in high iron, expressed as fold change relative to WT Mtb cultured in iron sufficient conditions. RNA extracted from biological triplicates was analyzed. The data are presented as means +/- SEM. **P≤ 0.01 ***P ≤ 0.001 (Student's t-test).
Figure 2A. MEV quantitation based on protein and lipid content in vesicle preparations isolated from the same number of Mtb cells treated with INH 50 ng.mlL-1 for 24 and 48 h relative to untreated cells. Three independent cultures were analyzed. Data are presented as mean ± SEM ***P ≤ 0.001 (Student's t-test).B. Immunodot blot assay detecting MEVs obtained from three independent cultures (1, 2, 3) and derived from the same number of Mtb exposed to 50 ng.mlL-1 INH for 24 and 48 h. A polyclonal antiserum raised against purified MEV (Prados-Rosales et al., 2014a) was used as a primary antibody.E. Protein and lipid content in vesicle preparations isolated from the same number of WT and Msm overexpressing iniACMtb (Msm iniACMtb). Three independent cultures were analyzed. Data are presented as mean ± SEM *P≤ 0.05; *** P≤ 0.001 (Student's t-test).H. The graph shows the avarage of nanoparticle size distribution in three independent vesicle preparations derived from WT (black) and Msm iniACMtb (grey).
Figure 3A. Quantitation of MEVs released by Mtb strains in low iron cultures based on vesicle protein and lipid content normalized to CFUs. Three biological replicates were processed. Data are presented as mean ± SD *P ≤0.05, **P≤ 0.005 (Student's t-test).C. Protein in vesicle isolates derived from three independent cultures of Mtb WT and iniA::hyg mutant grown in low (LI) or high iron (HI) MM with or without 50 ng·mL-1 INH. Data are presented as mean ± SEM *P ≤ 0.05. ***P≤ 0.001 (Student's t-test)D. Lipid in vesicle isolates derived from three independent cultures of Mtb WT and iniA::hyg mutant cultured in low (LI) or high iron (HI) MM with or without 50 ng·mL-1 INH. Data are presented as mean ± SD *P ≤ 0.05 (Student's t-test)